Hello everyone
I am trying to perform ChIP-seq experiments to find downstream effectors of a Transcription factor in Drosophila.
However, I am having trouble with the sonication steps. Since my final experiment needs to be done with a dissected tissue, I am trying to improve my protocol with S2 cells first.
The problem is that I didn't get any DNA from the 'size sample' (50ul extracted after sonication). After 1.8% formaldehyde incubation, sonication (in a volume of 2ml of RIPA buffer + EGTA, 30'ON/30'OFF for 7 to 10 cycles on ice), decrosslinking, RNase and PK treatment I run it in an agarose gel but there is nothing there. I tried to do it with 3.5 x 10^7 cells,. In other attempts I can see the DNA in the gel, but the smear fragment is too big, so the results I get are very irregular.
I have read in some protocols that the concentration of NaCl during decrosslinking is very important, but in others don't use it.
Could I get some advice of what to change in my protocol or what could it be happening?
Thank you very much!
Guillermo.
Guillermo,
We typically isolate chromatin before shearing, which entails detergent lysis of the cells followed by a low g spin to pellet the chromatin. The sonication conditions you listed seem mild. I would increase the number of cycles, also unless you are careful in how you place the tip of the sonicator in your sample you can get a large variation in shearing if it is not consistent. As for crosslinking, we routinely decouple overnight in TE with prot K at 56C and have seed no problems. I do not think NaCl is necessary.
Hi Bryan,
Thank you very much for your answer.
Now I am trying with genomic DNA (collecting around 5x10^7 S2 cells, fixating them in 1.8% FA for 20' and sonicating them), without Immunoprecipitation, in order to configure the correct shearing conditions. That's true that the sonication conditions could be mild. I next tried to do it with more cycles (5-10-20-25, sonicating in a 15% of maximum power, 30''ON/30''OFF each cycle, in a microson XL ultrasonic cell disruptor) and it doesn't seem to be sheared (picture attached). As you suggested, I will now try with more power in the sonicator, and see what happens.
Picture: different cycles of shearing: Lane 1, 5 cycles. Lane 2, 10 cycles. Lane 3, 20 cycles. Lane 4, 25 cycles, Lane 5, 25 cycles (rest of sample from lane 4). Lanes 1 and 2 got out of the wells when loading them in the gel, and fixed it in the next samples by adding 1ul of glycerol. In the 1 you can still see a very weak smear but without any size.
Thank you very much!
Best,
Guillermo.
We regularly disconnect chromatin before shearing, which involves cleanser lysis of the phones took after by a low g turn to pellet the chromatin. The sonication conditions you recorded appear to be mellow. I would build the quantity of cycles, likewise unless you are watchful by they way you put the tip of the sonicator in your example you can get an expansive variety in shearing in the event that it isn't steady. Concerning crosslinking, we routinely decouple overnight in TE with prot K at 56C and have seed no issues. I don't think NaCl is fundamental.
regards
Guillermo,
Since the lanes seem to be unevenly loaded it is a little hard to tell, but the DNA in lane 5 looks about as good as one can get with a sonicator. The majority of the DNA is sheared with a bulk of the DNA in the 300-500 bp range with a long smear going up. I have seen data where using freshly fixed cells that have not been previously frozen will help reduce the amount of smear and give a more uniform size. We tried this but it did not help us, but this could easily be cell type specific.
Good luck with your experiments
Bryan
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